Polycystic kidney disease (PKD) is an important cause of end-stage renal disease for which there is no proven effective therapy. We have found that cAMP levels are consistently increased in polycystic kidneys; that AVP V2R antagonism and somatostatin analogs lower these levels and ameliorate PKD in orthologous animal models, and that these two classes of drugs have additive protective effects on the kidney. These observations have led to clinical trials of V2R antagonists and somatostatin analogs with encouraging results. We have also found that the V2R agonist DDAVP exerts stronger cystogenic effects in PKD rat compared to mouse models, likely due to higher cAMP phosphodiesterase activity in mouse compared to rat kidneys; that development of PKD in Pkd2WS25/- is aggravated on a Pde1a, Pde1c or Pde3a, but not on Pde1b or Pde3b null backgrounds; that the cystogenic effect of DDAVP is enhanced on a Pde3a null background; and that morpholino induced knockdown of pde1a or pde3a induces a cystic phenotype in zebrafish embryos. The overall hypothesis of this grant application is that loss of polycystin function results in 1) dysregulation of intracellular Ca2+ dynamics causing upregulation of cAMP and protein kinase A signaling (due to inhibition of PDE1 and PDE3 activities and possibly activation of adenylyl cyclase 6) and defective purinergic signaling (due to inhibition of flow-induced ATP release), 2) increased sensitivity to AVP V2 receptor activation (due to inhibition of PDE1 and disruption of a purinergic negative feedback loop), and 3) maladaptive hyperphosphorylation of Ca2+ release channels in the endoplasmic reticulum (ER) leading to depletion of ER Ca2+ stores and reduced Ca2+ oscillations further aggravating the dysregulation of intracellular Ca2+ homeostasis. Specific aim 1 is to ascertain whether PDE1 and PDE3 isoforms redundantly regulate pools of cAMP that control cystogenesis (Hypothesis 1). Specific aim 2 is to ascertain whether purinergic signaling affects the development of PKD and whether downregulation of P2Y2 signaling enhances cystogenesis by disrupting a negative feed-back loop on V2 receptor signaling (Hypothesis 2). Specific Aim 3 is to use genetically engineered Ca2+, cAMP, and PKA biosensors, fluorescently labeled aquaporin-2 and ATP, and a phosphoproteomic approach to determine whether loss of polycystin reduces intracellular Ca2+ and alters PI3K/Akt signaling leading to increased cAMP levels, PKA-dependent hyperphosphorylation of ER Ca2+ channels, depletion of ER Ca2+ stores, increased mitochondrial Ca2+, reduced Ca2+ oscillations, and relative inhibition of exocytotic apical insertion of aquaporin-2 (AQP2) and vesicular exocytosis of ATP; and whether these effects will be enhanced by silencing the expression of Pde1a or Pde3a and by forskolin or DDAVP stimulation, and diminished by exogenous expression of Pde1a or Pde3a and by PKA inhibitors (Hypothesis 3). The results of these studies will inform on how Ca2+ and cAMP and PKA signaling are altered in PKD and may identify opportunities to enhance the efficacy of current investigational therapies targeting cAMP signaling in PKD.